Live Cell Protein Imaging of Tandem Complemented-GFP11-Tagged Coiled-Coil Domain-Containing Protein-124 Identifies this Factor in G3BP1-Induced Stress-Granules.

Coiled-coil domain-containing 124 protein is a multifunctional RNA-binding factor, and it was previously reported to interact with various biomolecular complexes localized at diverse subcellular locations, such as the ribosome, centrosome, midbody, and nucleoli. We aimed to better characterize the subcellular CCDC124 translocation by labelling this protein with a fluorescent tag, followed by laser scanning confocal microscopy methods. As traditional GFP-tagging of small proteins such as CCDC124 often faces limitations like potential structural perturbations of labeled proteins, and interference of the fluorescent-tag with their endogenous cellular functions, we aimed to label CCDC124 with the smallest possible split-GFP associated protein-tagging system (GFP11/GFP1-10) for better characterization of its subcellular localizations and its translocation dynamics. By recombinant DNA techniques we generated CCDC124-constructs labelled with either single of four tandem copies of GFP11 (GFP11 × 1::CCDC124, GFP11 × 4::CCDC124, or CCDC124::GFP11 × 4). We then cotransfected U2OS cells with these split-GFP constructs (GFP11 × 1(or X4)::CCDC124/GFP1-10) and analyzed subcellular localization of CCDC124 protein by laser scanning confocal microscopy. Tagging CCDC124 with four tandem copies of a 16-amino acid short GFP-derived peptide-tag (GFP11 × 4::CCDC124) allowed better characterization of the subcellular localization of CCDC124 protein in our model human bone osteosarcoma (U2OS) cells. Thus, by this novel methodology we successfully identified GFP11 × 4::CCDC124 molecules in G3BP1-overexpression induced stress-granules by live cell protein imaging for the first time. Our findings propose CCDC124 as a novel component of the stress granule which is a membraneless organelle involved in translational shut-down in response to cellular stress.

A multi-faceted approach to unravel coding and non-coding gene fusions and target chimeric proteins in ataxia.

Ataxia represents a heterogeneous group of neurodegenerative disorders characterized by a loss of balance and coordination, often resulting from mutations in genes vital for cerebellar function and maintenance. Recent advances in genomics have identified gene fusion events as critical contributors to various cancers and neurodegenerative diseases. However, their role in ataxia pathogenesis remains largely unexplored. Our study Hdelved into this possibility by analyzing RNA sequencing data from 1443 diverse samples, including cell and mouse models, patient samples, and healthy controls. We identified 7067 novel gene fusions, potentially pivotal in disease onset. These fusions, notably in-frame, could produce chimeric proteins, disrupt gene regulation, or introduce new functions. We observed conservation of specific amino acids at fusion breakpoints and identified potential aggregate formations in fusion proteins, known to contribute to ataxia. Through AI-based protein structure prediction, we identified topological changes in three high-confidence fusion proteins-TEN1-ACOX1, PEX14-NMNAT1, and ITPR1-GRID2-which could potentially alter their functions. Subsequent virtual drug screening identified several molecules and peptides with high-affinity binding to fusion sites. Molecular dynamics simulations confirmed the stability of these protein-ligand complexes at fusion breakpoints. Additionally, we explored the role of non-coding RNA fusions as miRNA sponges. One such fusion, RP11-547P4-FLJ33910, showed strong interaction with hsa-miR-504-5p, potentially acting as its sponge. This interaction correlated with the upregulation of hsa-miR-504-5p target genes, some previously linked to ataxia. In conclusion, our study unveils new aspects of gene fusions in ataxia, suggesting their significant role in pathogenesis and opening avenues for targeted therapeutic interventions.Communicated by Ramaswamy H. Sarma.

Dimerization underlies the aggregation propensity of intrinsically disordered coiled-coil domain-containing 124.

Coiled-coil domain-containing 124 (CCDC124) is a recently discovered ribosome-binding protein conserved in eukaryotes. CCDC124 has regulatory functions on the mediation of reversible ribosomal hibernation and translational recovery by direct attachment to large subunit ribosomal protein uL5, 25S rRNA backbone, and tRNA-binding P/A-site major groove. Moreover, it independently mediates cell division and cellular stress response by facilitating cytokinetic abscission and disulfide stress-dependent transcriptional regulation, respectively. However, the structural characterization and intracellular physiological status of CCDC124 remain unknown. In this study, we employed advanced in silico protein modeling and characterization tools to generate a native-like tertiary structure of CCDC124 and examine the disorder, low sequence complexity, and aggregation propensities, as well as high-order dimeric/oligomeric states. Subsequently, dimerization of CCDC124 was investigated with co-immunoprecipitation (CO-IP) analysis, immunostaining, and a recent live-cell protein-protein interaction method, bimolecular fluorescence complementation (BiFC). Results revealed CCDC124 as a highly disordered protein consisting of low complexity regions at the N-terminus and an aggregation sequence (151-IAVLSV-156) located in the middle region. Molecular docking and post-docking binding free energy analyses highlighted a potential involvement of V153 residue on the generation of high-order dimeric/oligomeric structures. Co-IP, immunostaining, and BiFC analyses were used to further confirm the dimeric state of CCDC124 predominantly localized at the cytoplasm. In conclusion, our findings revealed in silico structural characterization and in vivo subcellular physiological state of CCDC124, suggesting low-complexity regions located at the N-terminus of disordered CCDC124 may regulate the formation of aggregates or high-order dimeric/oligomeric states.

Facile modification of polycaprolactone nanofibers with egg white protein.

Synthetic polymers remain to be a major choice for scaffold fabrication due to their structural stability and mechanical strength. However, the lack of functional moieties limits their application for cell-based therapies which necessitate modification and functionalization. Blending synthetic polymers with natural components is a simple and effective way to achieve the desired biological properties for a scaffold. Herein, nanofibrous mats made of polycaprolactone (PCL) and egg white protein (EWP) blend were developed and further evaluated for use as a scaffold for tissue engineering applications. Homogeneous distribution of EWP was achieved throughout the nanofibrous mats, as shown by immunohistochemistry. ATR-FTIR analysis and contact angle measurements have further confirmed the presence of EWP on the surface of the samples. The swelling test showed that PCL/EWP nanofibers have higher water uptake than PCL nanofibrous mats. Also, EWP addition on the nanofibrous mats resulted in an increase in the tensile strength and Young's modulus of the mats, indicating that the presence of protein can greatly enhance the mechanical properties of the mats. A significantly higher, more uniform, and dispersed cell spreading was observed on days 7 and 14 than that on neat PCL mats, demonstrating the importance of providing the required cues for cell homing by the availability of EWP. Hence, EWP is shown to be a simple and low-cost source for the functionalization of PCL nanofibrous mats. EWP is, therefore, a facile candidate to enhance cellular interactions of synthetic polymers for a wide range of tissue engineering applications.

Identification of novel TUBB1 variants in patients with macrothrombocytopenia.

Background/aim: Macrothrombocytopenia is an autosomal-dominant disorder characterized by increased platelet size and a decreased number of circulating platelets. The membrane skeleton and the link between actin filaments of the skeleton and microtubules, which consist of alpha and beta tubulin [including the tubulin beta-1 chain (TUBB1)] heterodimers, are important for normal platelet morphology, and defects in these systems are associated with macrothrombocytopenia. Materials and methods: In this study, we sequenced the exons of the TUBB1 gene using DNA isolated from the peripheral blood samples of healthy controls (n = 47) and patients with macrothrombocytopenia (n = 37) from Turkey. The TUBB1 expression levels in fractioned blood samples from patients and healthy controls were analyzed by RT-qPCR and Western blot. Microtubule organization of the platelets in the peripheral blood smears of patients, and in mutant TUBB1-transfected HeLa cells, were analyzed by immunofluorescence staining. Results: A new TUBB1 c.803G>T (p.T178T) variant was detected in all of the control and patient samples. Importantly, we found 3 new heterozygous TUBB1 variants predicting amino acid substitutions: G146R (in 1 patient), E123Q (in 1 patient), and T274M (in 4 patients); the latter variant was associated with milder thrombocytopenia in cancer patients treated with paclitaxel. Ectopic expression of TUBB1 T274M/R307H variant in HeLa cells resulted in irregular microtubule organization. Conclusion: Further clinical and functional studies of the newly identified TUBB1 variants may offer important insights into their pathogenicity in macrothrombocytopenia.

The Thyroid Na+/I- Symporter: Molecular Characterization and Genomic Regulation.

Iodide (I-) is an essential constituent of the thyroid hormones triiodothyronine (T3) and thyroxine (T4), and the iodide concentrating mechanism of the thyroid gland is essential for the synthesis of these hormones. In addition, differential uptake of iodine isotopes (radioiodine) is a key modality for the diagnosis and therapy of thyroid cancer. The sodium dependent iodide transport activity of the thyroid gland is mainly attributed to the functional expression of the Na+/I- Symporter (NIS) localized at the basolateral membrane of thyrocytes. In this paper, we review and summarize current data on molecular characterization, on structure and function of NIS protein, as well as on the transcriptional regulation of NIS encoding gene in the thyroid gland. We also propose that a better and more precise understanding of NIS gene regulation at the molecular level in both healthy and malignant thyroid cells may lead to the identification of small molecule candidates. These could then be translated into clinical practice for better induction and more effective modulation of radioiodine uptake in dedifferentiated thyroid cancer cells and in their distant metastatic lesions.

Ligase-independent cloning of amylase gene from a local Bacillus subtilis isolate and biochemical characterization of the purified enzyme.

Five hundred ninety-seven bacterial isolates from Turkish hot spring water sources were screened for their ability to produce extracellular α-amylase. Among them, a high enzyme-producing Bacillus subtilis isolate, A28, was selected, and its α-amylase gene was cloned and expressed in Escherichia coli by a ligase-independent method. α-Amylase from the recombinant strain was purified to homogeneity by Q-Sepharose anion exchange and Sephacryl S-100 gel filtration chromatographies. The final yield of the enzyme was about 22.5 % of the initial activity, with a 16.4-fold increase in specific activity compared with the culture lysate. The optimum temperature and pH of the enzyme were 70 °C and 6.0, respectively. The enzyme was highly active at acidic-neutral pH range of 4.5-7.0. The amy28 α-amylase retained 100 % of its activity after incubation at 50 °C for 90 min. Co(+2), Cu(2+), Fe(2+), Fe(3+), Ni(+2), and Zn(+2) caused significant inhibition in enzyme activity, which was not affected by Na(+), Mg(2+), Li(+), and Ba(2+). The activity was inhibited about 70 % upon treatment of the enzyme with 10 mM ethylenediaminetetraacetic acid. However, Ca(2+) ions known as high temperature stabilizer for other amylases did not stimulate the activity of the enzyme. Due to pH stability and thermostability of the recombinant amylase, this enzyme may be suitable in starch processing, brewing, and food industries.

Improved activity and thermostability of Bacillus pumilus lipase by directed evolution.

To improve enzymatic activity of Bacillus pumilus lipases, DNA shuffling was applied to two lipase genes from local B. pumilus isolates. Using a high-throughput activity assay, the mutant with highest activity was selected. This chimeric mutant (L3-3), carrying two crossover positions and three point mutations, has a specific activity 6.4 and 8.2 times higher than the two parent enzymes. The mutant also is more tolerant to various detergents and organic solvents, and has a 9 times longer half-life at 50 °C. Homology modeling of mutant L3-3, based on the highly homologous B. subtilis lipase A, shows that the increased thermostability is likely due to structural rigidification and reduced surface hydrophobicity. Increased specific activity may result from the location of mutations close to the active site. Together, our results show that it is possible to evolve, by DNA shuffling, B. pumilus lipase variants with improved applicability as biocatalysts, even if the two parent enzymes are highly similar.